Project Summary The development of better cryopreservation methods and novel molecules with antifreeze properties is vital for the future of medicine and long term cryogenic storage of tissues, organs, and even whole organisms. This research project investigates the structure-activity relationship of a series of macrocycles with demonstrated antifreeze properties. The objective is to determine the minimal features critical for antifreeze properties in this structural class and thereby contribute to our long-term goal of a more detailed biophysical understanding of ice-growth inhibition at the molecular level of antifreeze compounds, including antifreeze proteins, in general. An exact mechanism of ice-binding and ice-growth inhibition by antifreeze molecules and materials at the molecular level is not fully understood, in spite of considerable efforts over the past decades, and this has hindered development of effective cryoprotection techniques and applications. Goals of the proposed plan will be accomplished in the following specific aims: Specific Aim 1 (SA1). Synthesis and purification of novel macrocycles. We will synthesize and purify a series of macrocycles based on an imidazole-4,5-dicarboxylic acid scaffold that varies the stereochemistry and functionality of two amino acids in the macrocycle, the N-alkyl group of a diamine linker, as well as the diamine linker length that are all part of the ring. The purified macrocycles will be initially characterized by combined LC-MS and NMR spectroscopy (1D and 2D, 1H and 13C). Specific Aim 2 (SA2). Structural and functional characterization of the novel macrocycles. We will use molecular modeling, NMR spectroscopy, and X-ray crystallography to determine the conformation(s) of the macrocycles. The antifreeze properties of the macrocycles along with the control compounds will be determined by a) ice recrystallization assays in both microcapillary tubes and the splat assay that starts with poly-nucleated ice wafers, b) thermal hysteresis measurements using differential scanning calorimetry (DSC), c) ice nucleation inhibition experiments, d) comparisons of ice morphology, as well as e) determining cryopreservation of E. coli BL21 cells as a way to demonstrate function in a model living system. Specific Aim 3 (SA3). Determination of minimal functional elements required for antifreeze activity. Our working hypothesis is that the structural (SA1) and conformational data (SA2) for the macrocycles will correlate to their antifreeze properties (SA2) and thereby allow us to define the minimal functional elements required for the active members of this series. In addition, we will use the ice nucleation and morphology data along with this structure-function information to postulate a mechanism of action for the active macrocycles, such as binding to a specific ice face.